DNA replication begins at multiple sites along the length of eukaryotic chromosomal DNA molecules. These sites are called origins. Considerable evidence suggests that origins are specific nucleotide sequences and that the same set of origins is used by all cells in a homogeneous cell population. A comparably large body of evidence suggests just the opposite - that the locations of origins may be determined by local chromatin structure or other factors, but not by nucleotide sequence. The experiments proposed here are designed to distinguish between these possibilities. We shall use two different experimental approaches. In both approaches we shall use the yeast, Saccharomyces cerevisiae, as our primary experimental organism because of its small genome size and because of the ready availability of cloned segments of DNA from a variety of chromosome regions. In the first approach we shall synchronize yeast cell populations to late G1 phase and then radiolabel the DNA made as the cells enter S phase. The labeled DNA will be digested by a restriction endonuclease, the restriction fragments will be separated from each other by 2D gel electrophoresis, and the labeled fragments will be identified by autoradiography. This approach provides information about a large number of restriction fragments simultaneously, but its resolving power is limited. For example, it is difficult to distinguish, using this approach, between origin-containing restriction fragments and adjacent non-origin-containing fragments. In our second approach we shall determine the direction(s) in which replication forks move through particular cloned segments of chromosomal DNA. From the direction(s) of replication fork movement we can infer the location(s) or origins. Thus this second approach provides more detailed information about a more limited region of the genome. Both approaches can be used to examine origin locations under a variety of physiological conditions which might be expected to alter chromatin structure. The combination of these two approaches should provide a complete answer to the question of whether specific nucleotide sequences serve as replication origins.